The present invention relates to engineering attenuated viruses by altering a non-coding region or the coding sequence of a viral nonstructural (NS) gene. In particular, the present invention relates to engineering live attenuated influenza viruses which induce interferon and related pathways. The present invention further relates to the use of the attenuated viruses and viral vectors against a broad range of pathogens and/or antigens, including tumor specific antigens. The present invention also relates to a host-restriction based selection system for the identification of genetically manipulated influenza viruses. In particular, the present invention relates to a selection system to identify influenza viruses which contain modified NS gene segments.
2.1. ATTENUATED VIRUSES
Inactivated virus vaccines are prepared by xe2x80x9ckillingxe2x80x9d the viral pathogen, e.g., by heat or formalin treatment, so that it is not capable of replication. Inactivated vaccines have limited utility because they do not provide long lasting immunity and, therefore, afford limited protection. An alternative approach for producing virus vaccines involves the use of attenuated live virus vaccines. Attenuated viruses are capable of replication but are not pathogenic, and, therefore, provide for longer lasting immunity and afford greater protection. However, the conventional methods for producing attenuated viruses involve the chance isolation of host range mutants, many of which are temperature sensitive; e.g., the virus is passaged through unnatural hosts, and progeny viruses which are immunogenic, yet not pathogenic, are selected.
Recombinant DNA technology and genetic engineering techniques, in theory, would afford a superior approach to producing an attenuated virus since specific mutations could be deliberately engineered into the viral genome. However, the genetic alterations required for attenuation of viruses are not known or predictable. In general, the attempts to use recombinant DNA technology to engineer viral vaccines have mostly been directed to the production of subunit vaccines which contain only the protein subunits of the pathogen involved in the immune response, expressed in recombinant viral vectors such as vaccinia virus or baculovirus. More recently, recombinant DNA techniques have been utilized in an attempt to produce herpes virus deletion mutants or polioviruses which mimic attenuated viruses found in nature or known host range mutants. Until very recently, the negative strand RNA viruses were not amenable to site-specific manipulation at all, and thus could not be genetically engineered.
2.2. THE INFLUENZA VIRUS
Virus families containing enveloped single-stranded RNA of the negative-sense genome are classified into groups having non-segmented genomes (Paramyxoviridae, Rhabdoviridae) or those having segmented genomes (Orthomyxoviridae, Bunyaviridae and Arenaviridae). The Orthomyxoviridae family, described in detail below, and used in the examples herein, contains only the viruses of influenza, types A, B and C.
The influenza virions consist of an internal ribonucleoprotein core (a helical nucleocapsid) containing the single-stranded RNA genome, and an outer lipoprotein envelope lined inside by a matrix protein (M). The segmented genome of influenza A consists of eight molecules (seven for influenza C) of linear, negative polarity, single-stranded RNAs which encode ten polypeptides, including: the RNA-directed RNA polymerase proteins (PB2, PB1 and PA) and nucleoprotein (NP) which form the nucleocapsid; the matrix proteins (M1, M2); two surface glycoproteins which project from the lipoprotein envelope: hemagglutinin (HA) and neuraminidase (NA); and nonstructural proteins whose function is unknown (NS1 and NS2). Transcription and replication of the genome takes place in the nucleus and assembly occurs via budding on the plasma membrane. The viruses can reassort genes during mixed infections.
Influenza virus adsorbs via HA to sialyloligosaccharides in cell membrane glycoproteins and glycolipids. Following endocytosis of the virion, a conformational change in the HA molecule occurs within the cellular endosome which facilitates membrane fusion, thus triggering uncoating. The nucleocapsid migrates to the nucleus where viral mRNA is transcribed as the essential initial event in infection. Viral mRNA is transcribed by a unique mechanism in which viral endonuclease cleaves the capped 5xe2x80x2-terminus from cellular heterologous mRNAs which then serve as primers for transcription of viral RNA templates by the viral transcriptase. Transcripts terminate at sites 15 to 22 bases from the ends of their templates, where oligo(U) sequences act as signals for the template-independent addition of poly(A) tracts. Of the eight viral mRNA molecules so produced, six are monocistronic messages that are translated directly into the proteins representing HA, NA, NP and the viral polymerase proteins, PB2, PB1 and PA. The other two transcripts undergo splicing, each yielding two mRNAs which are translated in different reading frames to produce M1, M2, NS1 and NS2. In other words, the eight viral mRNAs code for ten proteins: eight structural and two nonstructural. A summary of the genes of the influenza virus and their protein products is shown in Table I below.
The Influenza A genome contains eight segments of single-stranded RNA of negative polarity, coding for nine structural and one nonstructural proteins. The nonsructural protein NS1 is abundant in influenza virus infected cells, but has not been detected in virions. NS1 is a phosphoprotein found in the nucleus early during infection and also in the cytoplasm at later times of the viral cycle (King et al., 1975, Virology 64: 378). Studies with temperature-sensitive (ts) influenza mutants carrying lesions in the NS gene suggested that the NS1 protein is a transcriptional and post-transcriptional regulator of mechanisms by which the virus is able to inhibit host cell gene expression and to stimulate viral protein synthesis. Like many other proteins that regulate post-transcriptional processes, the NS1 protein interacts with specific RNA sequences and structures. The NS1 protein has been reported to bind to different RNA species including: vRNA, poly-A, U6, snRNA, 5xe2x80x2 untranslated region as of viral mRNAs and ds RNA (Qiu et al., 1995, Rna 1:304; Qiu et al., 1994, J. Virol. 68:2425). Expression of the NS1 protein from cDNA in transfected cells has been associated with several effects: inhibition of nucleo-cytoplasmic transport of mRNA, inhibition of pre-mRNA splicing, inhibition of host mRNA polyadenylation and stimulation of translation of viral mRNA (Fortes, et al., 1994, Embo J. 13:704; Enami, K. et al, 1994, J. Virol. 68: 1432 de la Luna, et al., 1995, J. Virol. 69:2427; Lu, Y. et al., 1994, Genes Dev. 8:1817; Park, et. al., 1995, J. Biol Chem. 270, 28433).
The present invention relates to genetically engineered live attenuated viruses which induce an interferon and related responses. In a preferred embodiment the present invention relates to engineering live attenuated influenza viruses which contain modified NS gene segments. The present invention also relates to both segmented and non-segmented viruses genetically engineered to have an attenuated phenotype and an interferon inducing phenotype, such a phenotype is achieved by targeting the viral gene product which interferes with the cellular interferon response. The attenuated viruses of the present invention may be engineered by altering the non-coding region of the NS gene segment that regulates transcription and/or replication of the viral gene so that it is down regulated. In non-segmented viruses, the down regulation of a viral gene can result in a decrease in the number of infectious virions produced during replication, so that the virus demonstrates attenuated characteristics. A second approach involves engineering alterations of the NS coding region so that the viral protein expressed is altered by the insertion, deletion or substitution of an amino acid residue or an epitope and an attenuated chimeric virus is produced. This approach may be applied to a number of different viruses and is advantageously used to engineer a negative strand RNA virus in which a NS gene product plays a role in regulating the interferon-mediated inhibition of translation of viral proteins.
The present invention is further related to vaccines and methods of inhibiting viral infection. The attenuated viruses of the present invention may be used to protect against viral infection. As demonstrated by the evidence presented in the Examples herein, the attenuated viruses of the present invention have anti-viral activity when administered prior to infection with wild-type virus, thus demonstrating the prophylactic utility of the attenuated viruses of the present invention.
The present invention is further related to a host-restriction based selection system for the identification of genetically manipulated influenza viruses. The selection system of the present invention is more particularly related to the identification of genetically manipulated influenza viruses which contain modified NS gene segments.
The present invention is based, in part, on the Applicants"" surprising discovery that an engineered influenza A virus deleted of the NS1 gene was able to grow in a cell line deficient in type 2 IFN production, but was undetectable in Madin-Darby canine kidney (MDCK) cells and in the allantoic membrane of embryonated chicken eggs, two conventional substrates for influenza virus. The Applicants"" further discovered that the infection of human cells with the engineered influenza A virus deleted of the NS1 gene, but not the wild-type virus, induced high levels of expression of genes under control of IFN-induced promoter. These results allow for the first time an efficient selection system for influenza viruses which contain NS1 mutants, where previously it was not possible to screen for viruses with an NS1 deleted phenotype.
The attenuated viruses of the invention may advantageously be used safely in live virus vaccine formulation. As used herein, the term xe2x80x9cattenuatedxe2x80x9d virus refers to a virus which is infectious but not pathogenic; or an infectious virus which may or may not be pathogenic, but which either produces defective particles during each round of replication or produces fewer progeny virions than does the corresponding wild type virus during replication. Pathogenic viruses which are engineered to produce defective particles or a reduced number of progeny virions are xe2x80x9cattenuatedxe2x80x9d in that even though the virus is capable of causing disease, the titers of virus obtained in a vaccinated individual will provide only subclinical levels of infection.